Maize is a key crop threatened by fall armyworm (FAW), leading to reliance on synthetic pesticides. This study explores maize flavonoids as a natural defense, using near isogenic lines (NILs) with varying flavonoid levels. High-flavonoid maize reduced FAW growth and survival, disrupting the insect’s gut microbiome. Axenic larvae did not exhibit flavonoid-induced mortality, highlighting a microbe-dependent defense mechanism. These findings support breeding maize for natural pest resistance, reducing pesticide dependence and environmental harm.